The present invention provides for a novel ligand that binds proteins belonging to the Eph subfamily of receptorlike protein tyrosine kinases, such as the Elk receptor and methods for making soluble forms of this ligand that are biologically active.
The ability of polypeptide ligands to bind cells and thereby elicit a phenotypic response such as cell growth, survival or differentiation is often mediated through transmembrane tyrosine kinases. The extracellular portion of each receptor tyrosine kinase (RTK) is generally the most distinctive portion of the molecule, as it provides the protein with its ligand-recognizing characteristic. Binding of a ligand to the extracellular domain results in signal transduction via an intracellular tyrosine kinase catalytic domain which transmits a biological signal to intracellular target proteins. The particular array of sequence motifs of this cytoplasmic, catalytic domain determines its access to potential kinase substrates (Mohammadi, et al.,1990, Mol. Cell. Biol., 11: 5068-5078; Fantl, et al., 1992, Cell, 69:413-413).
RTKs appear to undergo dimerization or some related conformational change following ligand binding (Schlessinger, J., 1988, Trend Biochem. Sci. 13:443-447; Ullrich and Schlessinger, 1990, Cell, 61:203-212; Schlessinger and Ullrich, 1992, Neuron 9:383-391); molecular interactions between dimerizing cytoplasmic domains lead to activation of kinase function. In some instances, such as the growth factor platelet derived growth factor (PDGF), the ligand is a dimer that binds two receptor molecules (Hart, et al., 1988, Science, 240: 1529-1531; Heldin, 1989, J. Biol. Chem. 264:8905-8912) while, for example, in the case of EGF, the ligand is a monomer (Weber, et al., 1984, J. Biol. Chem., 259:14631-14636).
The tissue distribution of a particular tyrosine kinase receptor within higher organisms provides relevant data as to the biological function of the receptor. The tyrosine kinase receptors for some growth and differentiation factors, such as fibroblast growth factor (FGF) are widely expressed and therefore appear to play some general role in tissue growth and maintenance. Members of the Trk RTK family (Glass and Yancopoulos, 1993, Trends in Cell Biol, 3:262-268) of receptors are more generally limited to cells of the nervous system, and the Nerve Growth Factor family consisting of NGF, BDNF, NT-3 and NT-4/5 (known as the neurotrophins) which bind these receptors promote the differentiation of diverse groups of neurons in the brain and periphery (Lindsay, R. M, 1993, in Neurotrophic Factors, S. E. Loughlin and J. H. Fallon, eds., pp. 257-284 (San Diego, Calif.: Academic Press). The localization of one such Trk family receptor, trkB, in tissue provided some insight into the potential biological role of this receptor, as well as the ligands that bind this receptor (referred to herein as cognates). Thus, for example, in adult mice, trkB was found to be preferentially expressed in brain tissue, although significant levels of trkB mRNAs were also observed in lung, muscle, and ovaries. Further, trkB transcripts were detected in mid and late gestation embryos. In situ hybridization analysis of 14 and 18 day old mouse embryos indicated that trkB transcripts were localized in the central and peripheral nervous systems, including brain, spinal cord, spinal and cranial ganglia, paravertebral trunk of the sympathetic nervous system and various innervation pathways, suggesting that the trkB gene product may be a receptor involved in neurogenesis and early neural development as well as play a role in the adult nervous system.
The cellular environment in which an RTK is expressed may influence the biological response exhibited upon binding of a ligand to the receptor. Thus, for example, when a neuronal cell expressing a Trk receptor is exposed to a neurotrophin which binds that receptor, neuronal survival and differentiation results. When the same receptor is expressed by a fibroblast, exposure to the neurotrophin results in proliferation of the fibroblast (Glass, et al., 1991, Cell 66:405-413). Thus, it appears that the extracellular domain provides the determining factor as to the ligand specificity, and once signal transduction is initiated the cellular environment will determine the phenotypic outcome of that signal transduction.
A number of RTK families have been identified based on sequence homologies in their intracellular domain. The receptor and signal transduction pathways utilized by NGF involves the product of the trk proto-oncogene (Kaplan et al., 1991, Nature 350:156-160; Klein et al., 1991, Cell 65:189-197). Klein et al. (1989, EMBO J. 8:3701-3709) reported the isolation of trkB, which encodes a second member of the tyrosine protein kinase family of receptors found to be highly related to the human trk protooncogene. TrkB binds and mediates the functional responses to BDNF, NT-4, and, to a lesser extent, NT-3 (Squinto, et al., 1991, Cell 65:885-903; Ip, et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:3060-3064; Klein, et al., 1992, Neuron, 8:947-956). At the amino acid level, the products of trk and trkB were found to share 57 percent homology in their extracellular regions, including 9 of the 11 cysteines present in trk. This homology was found to increase to 88 percent within their respective tyrosine kinase catalytic domains. The Trk gene family has now been expanded to include the trkC locus, with NT-3 having been identified as the preferred ligand for trkC (Lamballe, et al., 1991, Cell 66: 967-979; Valenzuela, et al. 1993, Neuron 10:963-974).
The Eph-related transmembrane tyrosine kinases comprise the largest known family of receptor-like tyrosine kinases, with many members displaying specific expression in the developing and adult nervous system. Two novel members of the Eph RTK family, termed Ehk (eph homology kinase) -1 and -2 were identified using a polymerase chain reaction (PCR)-based screen of genes expressed in brain (Maisonpierre, et al. 1993, Oncogene 8:3277-388). These genes appear to be expressed exclusively in the nervous system, with Ehk-1 expression beginning early in neural development. Recently, a new member of this group of related receptors, Ehk-3 has been cloned (Valenzuela, et al. 1995, Oncogene 10:1573-1580).
The elk gene encodes a receptorlike protein-tyrosine kinase that also belongs to the eph subfamily, and which is expressed almost exclusively in the brain (and at lower levels in the testes) (Letwin, et al. 1988; Oncogene 3:621-678; Lhotak, et al., 1991 Mol. Cell. Biol. 11: 2496-2502). Based on its expression profile, the Elk receptor and its cognate ligand are expected to play a role in cell to cell interactions in the nervous system. Other members of the Eph family of receptors that fall within the same subclass as Elk include the Nuk/Cek5, Hek2/Sek4 and Htk receptors (Brambilla and Klein, 1995, Mol. Cell. Neurosci., 6:487-495, Gale, et al., 1996, Neuron 17:9-19).
Unlike the Ehks and Elk receptors, the closely related Eck receptor appears to function in a more pleiotropic manner; it has been identified in neural, epithelial and skeletal tissues and it appears to be involved in the gastrulation, craniofacial, and limb bud sites of pattern formation in the mouse embryo (Ganju, et al. 1994, Oncogene 9:1613-1624).
The identification of a large number of receptor tyrosine kinases has far exceeded the identification of their cognate ligands. At best, determination of the tissues in which such receptors are expressed provides insight into the regulation of the growth, proliferation and regeneration of cells in target tissues. Because RTKs appear to mediate a number of important functions during development, their cognate ligands will inevitably play a crucial role in development.
Although a number of schemes have been devised for the identification of cognate ligands for the many orphan receptors that have been identified, very few such ligands have been identified, and the ligands that have been identified to date appear to have no activity other than the ability to bind their cognate receptor. For example, International Publication Number WO/94/11020 published on May 26, 1994 describes ligands that bind to the Eck receptor. In particular the ligand EBP (also known as B61) is described. However, although binding of B61 to the Eck receptor is disclosed, no biological activity is described. Similarly, despite the description in PCT Publication Number W094/11384 (published May 26, 1994) of a ligand that binds the Elk receptor, no biological activity was observed, regardless of whether the ligand was presented as membrane bound or in the form of an Fc dimer of the soluble ligand. With respect to the Elk receptor, however, chimeric EGFR-Elk receptors (having the extracellular domain of the EGFR fused to the Elk cytoplasmic domain) have been used to demonstrate the functional integrity (as measured by EGF-stimulated autophosphorylation) of the enzymatic domain of this receptor. (Lhotak and Pawson, 1993, Mol. Cell. Biol. 13:7071-7079).
The present invention, provides for a novel polypeptide ligand, designated as Efl-6, that binds to the Elk, Nuk/Cek5, Hek2/Sek4, Htk, and Sek1 receptors on cells. More importantly, the invention provides a means of making biologically active, soluble forms of this ligand, which are useful in promoting a differential function and/or influencing the phenotype, such as growth and/or proliferation, of receptor bearing cells. The invention also provides for nucleic acids encoding such polypeptide ligands, and both prokaryotic and eukaryotic expression systems for producing such proteins. The invention also provides for antibodies to these ligands.
According to the invention, soluble forms of the ligands described herein may be used to promote biological responses in Elk, Nuk/Cek5, Hek2/Sek4, Htk, and Sek1 receptor-expressing cells. In particular, a general method is described herein which produces xe2x80x9cclusteringxe2x80x9d of ligands for eph-related receptors, which functions to make otherwise inactive soluble ligands biologically active, or which enhances the biological activity of ligands that, absent such clustering, would have only low levels of biological activity.
The ligands described herein also have diagnostic utilities. In particular embodiments of !the invention, methods of detecting aberrancies in their function or expression may be used in the diagnosis of neurological or other disorders.
In other embodiments, manipulation of the interaction between the ligands and their cognate receptor may be used in assay systems designed to identify both agonists and antagonists of Eph receptor ligands. Such agonists and antagonists may be developed for use in the eventual treatment of neurological or other disorders.